Background: Stomata analysis using microscope imagery provides important insight into plant physiology, health and the surrounding environmental conditions. Plant scientists are now able to conduct automated high-throughput analysis of stomata in microscope data, however, existing detection methods are sensitive to the appearance of stomata in the training images, thereby limiting general applicability. In addition, existing methods only generate bounding-boxes around detected stomata, which require users to implement additional image processing steps to study stomata morphology. In this paper, we develop a fully automated, robust stomata detection algorithm which can also identify individual stomata boundaries regardless of the plant species, sample collection method, imaging technique and magnification level.Results: The proposed solution consists of three stages. First, the input image is pre-processed to remove any colour space biases occurring from different sample collection and imaging techniques. Then, a Mask R-CNN is applied to estimate individual stomata boundaries. The feature pyramid network embedded in the Mask R-CNN is utilised to identify stomata at different scales. Finally, a statistical filter is implemented at the Mask R-CNN output to reduce the number of false positive generated by the network. The algorithm was tested using 16 datasets from 12 sources, containing over 60,000 stomata. For the first time in this domain, the proposed solution was tested against 7 microscope datasets never seen by the algorithm to show the generalisability of the solution. Results indicated that the proposed approach can detect stomata with a precision, recall, and F-score of 95.10%, 83.34%, and 88.61%, respectively. A separate test conducted by comparing estimated stomata boundary values with manually measured data showed that the proposed method has an IoU score of 0.70; a 7% improvement over the bounding-box approach.Conclusions: The proposed method shows robust performance across multiple microscope image datasets of different quality and scale. This generalised stomata detection algorithm allows plant scientists to conduct stomata analysis whilst eliminating the need to re-label and re-train for each new dataset. The open-source code shared with this project can be directly deployed in Google Colab or any other Tensorflow environment.